Family of Hedgehog genes have been studied from the beginning of 1990th years when the first gene of this kind has been found at drosophila-fly. Drosophila's maggots exhibiting the mutant version of this gene are covered with sharp excrescences looking like hedgehog needles (from here the gene name hedgehog—a hedgehog). These genes code Hedgehog (Hh) secreted proteins, represented at vertebrated animals by Desert (DHh), Sonic (SHh) and Indian (IHh) Hh proteins. When entering Hh signalling system Hh proteins are subjected to intramolecular splitting and lipidic modification, the latter is catalyzed by C-terminal fragment of protein-precursor acting as cholesterol transferase [Porter J. A., Young K. E., Beachy P. A. Cholesterol modification of hedgehog signalling proteins in animal development. Science. 1996; 274: 1597]. As a result of it a peptide with mass about 19 kD esterified at C-termination with cholesterol molecule (HhNp) is formed. This modified peptide is responsible for all known signalling activities of Hh proteins. At mammals SHhNp-protein is subjected to further palmitoylation at N-termination that leads to enhancement of its signalling activity. By means of Hh signalling system Hh proteins regulate the process of various organs and tissues construction in the course of embryonal development [Neumann C. J. Hedgehogs as negative regulators of the cell cycle. Cell Cycle. 2005; 4(9):1139-40]. For example, SHh, among other things, is connected with morphogenesis of nervous tissue and intestinal tract, and DHh is involved in differentiation of male genital organs. At healthy adult individuals these proteins are operated by stem cells [Lewis M. T., Visbal A. P. The hedgehog signalling network, mammary stem cells, and breast cancer: connections and controversies. Ernst. Schering Found. Symp. Proc. 2006; 5:181-21]. The distortion of Hh signalling system, in particular, its enhanced activity leads to cancerous transformation of tissue [Evangelista M., Tian H., de Sauvage F. J. The hedgehog signalling pathway in cancer. Clin. Cancer Res. 2006; 12(20 Pt 1):5924-8]. A simplified model of Hh signalling system including the key components and biotargets in Hh signalling system is described in: [Kiselyov A. S., Tkachenko S. E., Balakin K. V., Ivachtchenko A. V. Small-molecule modulators of Hh and Wnt signalling pathways. Expert Opin. Ther. Targets. 2007; 11(8): 1087-1101].
Hh proteins transfer a signal into the cell by means of two transmembrane proteins: suppressor Ptc and protooncogenous Smo. Hh protein is directly linked with 12-tuple membrane penetrating protein Ptc, which is negative Smo protein modulator. Smo protein belonging to serpentine receptor family, penetrates cell membrane seven times and contains N-terminal CRD domain (cysteine-rich domain). There is some similarity of protein Smo to 7-transmembrane receptors connected with G-proteins, however, up to now neither endogenous Smo-modulator, nor corresponding effector G-protein have been found.
In the absence of Hh-peptide Ptc Protein completely inactivates Smo receptor and Hh signalling system does not operate. There are some possible ways of Ptc and Smo proteins interaction [Ogden S. K., Ascano M., Stegman M. A., Robbins D. J. Regulation of Hedgehog signaling: a complex story. Biochem. Pharmacol. 2004; 67(5):805-14], in particular, it is supposed the formation of heterodimeric receptor. However, on the basis of experimentally established difference in membrane localizations of these proteins in vivo alternative catalytical models of their interaction were offered.
Not so much is known about the mechanism of action of the molecules participating in the further formation of SHhNp signalling cascade in cytosol. Signalling complex comprising transcriptional factors Gli1, Gli2 or Cli3 (homologues of Cubitus Interruptus (Ci) at flies Drosophila), is regulated by physical interaction with microtubules. Serine-threonine kinase Fu (Fused), suppressor Fu (SuFu) and kinesin-alike protein Cos 2 are the components of this complex [Claret S., Sanial M., Plessis A. Evidence for a novel feedback loop in the Hedgehog pathway involving Smoothened and Fused. Cur.r Biol. 2007; 17(15): 1326-33]. In the absence of signal transmission from Hh protein the said components of the complex are associated with microtubules. It is supposed, that the essential role of Fu kinase consists in directing suppressor and Cos 2 protein functions. Kinase Fu mutation leads to the loss of any form of Ci processing, that, eventually, initiates stopping of Hh signalling. It is expected that Fu kinase could be a perspective medicinal biotarget, and its inhibiting will cause blockade of Hh signalling system. At the same time, the mechanism of Fu kinase activation is not entirely clarified, and its analogue at mammals has not been determined yet.
Thus, SHhNp-protein induces phosphorylation and dissociation of signalling complex from the surface of microtubules. This facilitates translocating of transcriptional factors Gli1, Gli2 and/or Cli3 into cell nucleus. There they associate with c-AMP-sensitive binding protein (CBP) and initiate transcription, that leads, among other processes, to enhancement of activity of alkaline phosphatase [Pepinsky R. B., Zeng C. Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem. 1998; 273: 14037-45] and cellular proliferation [Chen C. H., Von Kessler D. P., Park W., Wang B., Ma Y., Beachy P. A: Nuclear trafficking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression. Cell. 1999; 98(3): 305-16]. It is supposed, that Gli3 carries out regulatory functions at warm-blooded, while Gli1 and Gli2 are activators [Park H. L., Bai C., Platt K. A. Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation. Development. 2000 127(8): 1593-605].
It is also supposed that some biological processes and biotargets in Hh-signalling cascade could be sensitive to the action of small molecules and could be controlled by them, the verity of this is supported by the whole series of drug candidates, being at present at various stages of clinical investigation (Table 1, only signalling cascade antagonists are represented) [http://www.integrity.prous.com].
TABLE 1Hh-signalling cascade modulators at various stages of clinical investigation(data for november 2007)TherapeuticCompoundMechanismPhase of testSponsorgroupCUR-61414Smo receptorPhase IRoche, Curis,oncology(G-024856)antagonist;GenentechbasalHh-signalling cascadecarcinomainhibitorCyclopamineSmo receptorPreclinicalJohns Hopkinsoncologyantagonist, HhphaseUniversity, Howardsignalling inhibitorHughes MedicalInstitute, CurisHhAntagSmo receptorPreclinicalCurisoncologyantagonist;phase/Hh-signalling cascadePhase IinhibitorSANT-1Smo receptorPreclinicalJohns Hopkinsoncologyantagonist;phaseUniversityHh-signalling cascadeinhibitorCUR-691Smo receptor,PreclinicalCuris, Genentechoncologyantagonist;phaseBrainHh-signalling cascadetumoursinhibitorIPI-609Hh-signalling cascadePreclinicalInfinityoncologyinhibitorphasePharmaceuticalsIPI-926Hh-signalling cascadePreclinicalInfinityoncologyinhibitorphasePharmaceuticalsNSC-75503Hh-signalling cascadePreclinicalKarolinska InstituteoncologyinhibitorphaseGANT-61Hh-signalling cascadePreclinicalKarolinska InstituteoncologyinhibitorphaseMEDI-562Hh-signalling cascadePreclinicalMedImmuneoncologyinhibitorphase
In the context of novel drugs creation protein Smo in this cascade is considered to be one of the most perspective, and that was confirmed by the discovery of various, structurally similar ligands [Frank-Kamenetsky M., Zhang X. M., Bottega S. Small-molecule modulators of Hedgehog signaling: Identification and characterization of smoothened agonists and antagonists. J. Biol. 2002; 1(2): 10].
Steroid teratogenic alkaloides cyclopamin and jervine, isolated from poisonous Hellebore (Veratrum californicum), are the first discovered small molecule antagonists of Hh-signalling cascade [Williams J. A., Guicherit O. M., Zaharian B. I. Identification of a small molecule inhibitor of the hedgehog signalling pathway: effects on basal cell carcinoma-like lesions. Proc. Natl. Acad. Sci. 2003; 100(8): 4616-21]. Cyclopamin inhibits Smo functions, directly binding to heptahelical protein and preserving by that its inactive conformation [Taipale J., Chen J. K., Cooper M. K. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature. 2000; 406(6799): 1005-9]. Cyclopamin shows high antitumor activity in vitro and in vivo in models of medulloblastoma [Berman D. M., Karhadkar S. S., Hallahan A. R. Medulloblastoma growth inhibition by hedgehog pathway blockade. Science. 2002; 297(5586): 1559-61], basal carcinoma [Williams J. A. Hedgehog signaling pathway as a target for therapeutic intervention in basal cell carcinoma. Drug News Perspect. 2003; 16(10): 657-62] and breast cancer, in which Hh-signalling cascade is activated [Kubo M., Nakamura M., Tasaki A. Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer. Cancer Research. 2004; 64(17): 6071-6074]. It is also possible, that arrest of Hh-signalling cascade induced by cyclopamin could be used as basically novel approach for treatment of psoriasis [Meth M. J., Weinberg J. M. Cyclopamine: inhibiting hedgehog in the treatment of psoriasis. Cutis. 2006; 78(3):185-8].
Abberant activation of Hh-signalling cascade has been found in various tumours of humans [Evangelista M., Hua Tian H., de Sauvage F. J. The Hedgehog Signaling Pathway in Cancer Clin Cancer Res. 2006; 12: 5924-5928], in particular, for the most aggressive forms of prostate cancer [Aparicio A. L. M., Campelo G. R., Espinosa C. J., Ayerbes V. M., Lopez R. M., Prado D. S., Gallego A. G. Prostate cancer and Hedgehog signalling pathway. Clin. Transl. Oncol. 2007; 9(7): 420-8]. That is the reason for the high interest to antagonists of this cascade as to essentially novel low-toxic oncolytic drug substances.
Thus, searching for effective inhibitors of Hh-signalling cascade is an extremely important, urgent and promising problem.
For the purpose of finding out novel highly effective oncolytic drug substances the authors of the invention carried out a broad screening investigation of compounds with low-molecular weight of different structures among which hit-compounds and leader-compounds belonging to eight families of heterocyclic compounds have been found, directed modifications of many positions of discovered structures were performed, and as a result of all that series of novel derivatives of the said heterocycles have been synthesized which are original and highly effective inhibitors of Hh signalling cascade. The prepared physiologically active heterocyclic compounds include derivatives of 2,6-dihydro-7H-pyrazolo[3,4-d]pyridazin-7-one and 1,4-dihydropyrazolo[3,4-b]thiazin-5(6H)-one; N-acylated 4-imidazo[1,2-a]pyridin-2-yl- and 4-imidazo[1,2-a]pyrimidin-2-yl-anilines; amides of [(4H-thieno[3,2-b]pyrrol-5-yl)carbonyl]piperidine-4-carboxylic acid; amides of 2-(4-carbamoylpiperidin-1-yl)isonicotinic acid; amides of N-sulfonyl-1,2,3,4-tetrahydroquinoline-6-carboxylic acid; and N-acylated 3-azolyl derivatives of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridine as well. The prepared compounds have not been described before; their ability to inhibit Hh signalling cascade has not been known either.